A new method for obtaining bankable and expandable adult-like microglia in mice

Background The emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia. We aim to develop a new method for obtaining bankable and expandable adult-like microglia in mice. Methods The head neuroepithelial layer (NEL) that composed of microglial progenitor and neuroepithelial cells at mouse E13.5 was dissected and then cultured or banked. Microglia (MG) isolated from the cultured NEL by magnetic-activated cell sorting system were obtained and named NEL-MG. Results The NEL included microglia progenitors that proliferate and ramify over time with neuroepithelial cells as feeder. In functional analysis, NEL-MG exhibited microglial functions, such as phagocytosis (microbeads, amyloid β, synaptosome), migration, and inflammatory response following lipopolysaccharide (LPS) stimulation. NEL was passage cultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term passage culture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. NEL-MG could be re-expandable when they were plated again on neuroepithelial cells. Conclusions This new method effectively contributes to obtaining sufficient matured form of microglia (adult-like microglia), even when only a small number of experimental animals are available, leading to a broad application in the field of neuroscience. Supplementary Information The online version contains supplementary material available at 10.1186/s12974-021-02351-4.


Introduction
Since their discovery in the twentieth century by Rio-Hortega, microglia were considered professional phagocytes, similar to macrophages for a long time. However, recent studies propose microglia as a promising target beyond phagocytes under neuroinflammatory/neurodegenerative conditions [1]. Microglia are capable You et al. Journal of Neuroinflammation (2021) 18:294 cell line and primary culture using neonatal cortex or the adult brain. Microglial cell lines, such as BV2, N9, SIM-A9, Mocha, and MHC3, have an advantage in proliferation and passage culture; however, they differ from adult microglia in genetic and functional aspects due to immortalization [6]. Neonatal microglia have not been artificially manipulated, but have disadvantages regarding their proliferative capacity, and passage culture and banking abilities. Both the microglial cell line and neonatal microglia rarely express key adult microglia genes; in particular, TMEM119 immunoreactivity (IR) is missed [7,8] because microglial cells develop according to a stepwise program [4]. Acute isolation of adult microglia is challenging due to their restricted proliferative capacity, cell viability, and requirement of a greater number of animals [6,9]. Granulocyte macrophage colony stimulating factor (GM-CSF) and microglia growth supplement (Scien-Cell) might allow postmortem adult human microglia to proliferate in culture to high passage without several phenotypes alteration [10], but it requires further validation on the microglial signature genes. The iPSC-derived microglia-like cells might also be an alternative method; however, this method is expensive, time-consuming, and not easily accessible [11].
In addition, iPSC-derived microglia-like cells do not originate in the yolk sac and do not show adequate TMEM119 IR compared to adult microglia [12]. Thus, while the demand for microglia for use in research is increasing, there are no current in vitro methodologies that overcome the above-mentioned limitations. Mouse microglia progenitors begin to migrate from the yolk sac into the CNS at E8.5 and surround the neuroepithelial layer (NEL) in the part corresponding to the head [1,13]. Subsequently, mouse microglia progenitors move into the CNS parenchyma until approximately E18.5 and are matured in the CNS microenvironment [14]. As the CNS matures, microglia acquire a ramified morphology, surveying the surrounding parenchyma via movement of dynamic processes [2].
In the support of the idea that microglia progenitor derived from yolk sac should pass NEL to enter CNS parenchyma, we could obtain and co-culture microglial progenitors and neuroepithelial cells together by dissecting mouse E13.5 head NEL, expecting that neuroepithelial cells play a role as feeder cells. CD11bpositive cells were isolated from cultured NEL by magnetic-activated cell sorting (MACS) system and they were named NEL-MG. By this new methodology, we could establish in vitro approach to obtain bankable and expandable matured form of microglia (adult-like microglia).

Ethics approval and consent to participate
All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the CHA University (IACUC200116).

Mouse embryonic neuroepithelial layer dissection, culture, and banking
Uteri from pregnant mice (female C57BL/6, Orient Bio Inc. Seoul, Korea or female B6-EGFP mice, Jackson lab) were dissected and soaked in Hanks' Balanced Salt Solution (HBSS, Invitrogen, USA). The umbilical cord and yolk sac in the mesometrial surface of the uterus were removed using microsurgical instruments under a microscope and embryos were taken out of the uterus. Then, the NEL was dissected carefully using a pair of microsurgical scissors. The shredded tissue was incubated with 1 mL of 1X Trypsin-EDTA (ThermoFisher, USA) for 3 min at 37 °C in a water bath. After centrifuging at 300 × g for 5 min, the cells were plated onto 25 cm 2 flasks coated with poly-Dlysine (Sigma, MO, USA). The cells were cultured in DMEM high glucose medium containing 10% FBS, 1X penicillin:streptomycin and 0.1% GlutaMAX at 37 °C in a 5% CO 2 incubator. The culture medium was replaced with 5 mL of fresh growth medium after 24 h. Subsequently, half the culture medium volume was replaced with an equal volume of fresh growth medium twice per week. For banking, NEL were frozen and stored for about ten months in freezing media composed of DMEM with 20% FBS and 10% DMSO in liquid nitrogen (about − 196 °C). For thawing, the stock vials were dissolved at 37 °C water bath for 3 min and diluted with culture medium.

Isolation of microglial cells from NEL
On ~ day 21, when stratification was reached, NEL culture flasks were incubated with 1X trypsin-EDTA for 1 min, resulting in the detachment of an intact layer of cells in pieces. The pellet was resuspended in cold MACS buffer (containing a 1-volume dilution of PBS, 2 mM EDTA, and 0.5% BSA, pH 7.2), and myelin removal beads (Myelin Removal Beads II, 130-96-733, Miltenyi Biotec) were used according to the manufacturer's protocol . Briefly, cells were incubated with the microbeads coupled anti-CD11b mAb at 4 °C for 15 min, and then the cells were washed and resuspended in 500 µL PB buffer. The magnetically labeled cells were separated by MS column on a magnetic separator. The column was washed thrice with PBS buffer, and the separated cells (NEL-MG) were obtained via positive selection. NEL-MG were resuspended in microglial complete culture medium (DMEM, 10% FBS, 0.1% GlutaMAX, 5 μg/mL insulin, and 1% penicillin/streptomycin), transferred to poly-D-lysine-coated plates at a density of 2.5 × 10 5 cells/mL, and cultured for subsequent molecular studies.

Adult microglial cell isolation
Adult microglial cells were isolated from 8 to 10 weekold mice (male C57BL/6, Orient Bio Inc. Seoul, Korea). Enzymatic cell dissociation was performed using an Adult Brain Dissociation Kit (130-107-677, Miltenyi Biotec) according to the manufacturer's instructions. Brain tissue pieces (up to 500 mg) were transferred into a gen-tleMACS C tube (130-096-334, Miltenyi Biotec) containing 1950 μL of enzyme mix 1 (enzyme P and buffer Z), and then 30 μL of enzyme mix 2 (enzyme A and buffer Y) was added. The gentleMACS C tube was tightly closed and attached upside down onto the sleeve of the gen-tleMACS Octo Dissociator with Heaters (130-096-427, Miltenyi Biotec), and the appropriate gentleMACS program (37C_ABDK_01) was run. After brief centrifugation to collect the samples at the bottom of the tube, the samples were filtered through a 70 μm strainer (130-098-462, Miltenyi Biotec), washed with D-PBS, and centrifuged again. The pellet was resuspended in cold D-PBS. The myelin and cell debris were removed using debris removal solution, followed by subsequent removal of erythrocytes using a red blood cell removal solution. Pure adult microglial cells were magnetically isolated using microbeads-coupled anti-CD11b mAb, as stated previously.

Neonatal cortical microglia culture
Postnatal 1-to 2-day-old B6 mice (Orient Bio Inc. Seoul, Korea) were killed and soaked in 75% ethanol for 1 min. The cerebral hemispheres were dissected following standard techniques and anatomical landmarks, and the meninges were peeled off. The hippocampus, basal ganglion, and olfactory bulb were carefully removed using microsurgical instruments under a microscope, and the remaining cortical tissue was minced using a pair of microsurgical scissors. The shredded tissue was then incubated with 3 mL of HBSS (Invitrogen) for 5 min at 37 °C in a water bath. After centrifuging at 300 × g for 5 min, the cells were plated onto 25 cm 2 flasks coated with poly-D-lysine (Sigma). Mixed glial cells were cultured in DMEM high glucose medium containing 10% FBS and 0.1% GlutaMAX at 37 °C and 5% CO 2 in an incubator. The culture medium was replaced with 5 mL of fresh growth medium after 24 h. Subsequently, half of the culture medium volume was replaced with an equal volume of fresh growth medium twice a week. Stratification was reached at the end of this period, and the microglial cells in the upper layer were harvested. On day 14, flasks were incubated with 1X trypsin-EDTA for 1 min, resulting in the detachment of an intact layer of cells in one piece. The pellet was resuspended in cold MACS buffer (containing 1-volume dilution of PBS, 2 mM EDTA, and 0.5% BSA, pH 7.2), and then myelin removal beads (Myelin Removal Beads II, 130-96-733, Miltenyi Biotec) were used according to the manufacturer's protocol. Neonatal microglia were isolated with the microbeads coupled anti-CD11b mAb b MACS system. The isolated microglia were resuspended in microglial complete culture medium (DMEM, 10% FBS, 0.1% GlutaMAX, 5 μg/mL insulin, and 1% penicillin/streptomycin) and transferred to poly-D-lysine-coated plates at a density of 2.5 × 10 5 cells/mL for molecular studies.

Flow cytometry
The cells were collected and labeled using fluorochromeconjugated monoclonal antibodies recognizing antigens (CD11b-PE, BD Biosciences #553311) at 4 °C for 15 min. After labeling, the cells were washed twice in PBS and resuspended in a final volume of 400 μL. Flow cytometry was performed using a CytoFLEX (Beckman Coulter, cytoflex B4-R1-Vo) and the data were analyzed using CytExpert software.

Immunocytochemistry
Cells were seeded on glass cover slips in 24-well plates. Cells were washed with PBS and cultured; the cultured cells were then fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100 for 5-10 min. Indirect immunofluorescence was performed using the following primary antibodies: . Cells were incubated with the primary antibodies diluted in 0.5% Triton X-100 in PBS containing 5% normal donkey serum at 4 °C overnight. After rinsing thrice with PBS for 5 min, Alexa 488-or Alexa-594-conjugated secondary antibodies (Abcam) were used for detection. Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI; Sigma). Cells without the addition of primary antibodies served as negative controls. Fluorescent images were taken using a confocal microscope (LSM 700, Carl Zeiss, Jena, Germany).

Purification and labeling of synaptosome
One hemisphere (male C57BL/6, Orient Bio Inc. Seoul, Korea), excluding the cerebellum, (~ 200-400 mg), was homogenized in 10 volumes of Syn-PER Synaptic Protein Extraction Reagent (Thermo Fisher Scientific, Part No. 87785) using a 7 mL Dounce tissue grinder with 10 upand-down even strokes. The homogenate was centrifuged at 1200 × g for 10 min to remove the cell debris, and the supernatant was centrifuged at 15,000 × g for 20 min to obtain the synaptosome pellet. The pellets were gently resuspended in the respective reagent. Synaptosomes were conjugated with an amine-reactive dye (pHrodo Red, SE; Thermo Scientific; #P36600) in 0.1 M sodium carbonate (pH 9.0) at room temperature. After 2 h of incubation, unbound pHrodo was washed-out via multiple rounds of centrifugation and pHrodo-conjugated synaptosomes were resuspended in isotonic buffer containing 5% DMSO for subsequent freezing (Invitrogen, #P36600).

Scratch wound assay
NEL-MG were seeded onto 24-well plates in a 100% confluent monolayer until they were 95% confluent and were wounded by making a perpendicular scratch with a 200 μL pipette tip. The cells were replenished with fresh growth medium and wound closure was documented by photographing the same region at different times (0-6 h). The wound area was calculated as the open wound area/ total area.

Cytokine profiles
The supernatant was analyzed using a Proteome Profiler Mouse Cytokine Array Panel A Kit (R&D Systems; catalog number ARY006) at the baseline and after LPS stimuli according to the manufacturer's indications. Images were captured using a LAS 4000 (lmageQuant ™ ) and analyzed using ImageJ software program.

RNA sequence and data analysis
RNA quality was assessed with an Agilent 2100 bioanalyzer using an RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, Netherlands). The library construction was performed using a QuantSeq 3′-mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer's instructions. High-throughput sequencing was performed as 75 single-end sequences using Next-Seq 500 (Illumina, Inc., USA). QuantSeq 3′-mRNA-Seq reads were aligned using Bowtie2 (Langmead and Salzberg 2012). Differentially expressed genes were determined based on the counts from unique and multiple alignments using coverage in Bedtools (Quinlan AR, 2010). The Read Count data were processed based on the quantile normalization method using Edge R within R (R Development Core Team 2016) using Bioconductor (Gentleman et al. 2004). Gene classification was based on searches done using DAVID (http:// david. abcc. ncifc rf. gov/) and Medline databases (http:// www. ncbi. nlm. nih. gov/).

CellTracker labeling
NEL-MG were labeled using CellTrackerRed CMTPX (Invitrogen) before mixing with NS. Labeling was performed according to the manufacturer's indications.

Statistical analysis
The statistical significance of differences between groups was assessed using an unpaired t-test or one-way analysis of variance using GraphPad Prism version 7 for Mac (GraphPad, La Jolla, CA). A post hoc test was performed using one-way analysis of variance when the p-values were significant (p < 0.05).

Expandable and ramified microglial cells are generated from mouse E13.5 head neuroepithelial layer
To determine the optimal period for obtaining a high yield of microglia progenitors, we dissected head NEL from mouse E9.5, E13.5, and E17.5 and cultured them (Fig. 1A). Immunofluorescence analyses revealed that the highest numbers of microglia progenitors were obtained from the mouse E13.5 NEL compared to that of mouse E9.5 or E17.5 (Additional file 1: Fig. S1). The flow cytometry results showed that E13.5 NEL contain more CD11b-positive cells than the brain cortex at E13.5 (Additional file 1: Fig. S1). We next examined whether microglia progenitors were expandable and ramified over time in this culture system. The proportion of CD11b-positive cells increased from 14.2 up to 54.25% after 21 days of culture (Fig. 1B). The immunofluorescence results showed that the number of microglial cells that were IBA-1-, PU1-, and F4/80-positive increased over time. The percentage of double-positive cells (Ki67 and IBA-1) also increased to 50%, suggesting that microglia have a high proliferative capacity in this culture system (Fig. 1C).

Functional validation of magnetically isolated CD11b-positive cells (NEL-MG)
CD11b-positive cells were isolated using a MACS system from the E13.5 NEL (NEL-MG). NEL-MG stained positive for microglia markers with IBA-1 and CX3CR1. NEL-MG showed a relatively weak TMEM119 IR; however, they did not express Ki67, indicating that NEL-MG alone did not proliferate without neuroepithelial cells ( Fig. 2A). Next, we conducted microglia-specific functional assays, including phagocytosis, migration, and cytokine/chemokine release using NEL-MG. NEL-MG showed phagocytic function against stimuli, such as synaptosome, amyloid β, and microbeads (Fig. 2B). In addition, NEL-MG had higher phagocytic activity than BV2, neonatal microglia, and adult microglia (Additional file 1: Fig. S2). The scratch wound assay results showed that NEL-MG could migrate (Fig. 2C). Based on the cytokine and chemokine profiles, unstimulated NEL-MG released detectable cytokines and chemokines, including the C-X-C motif chemokine ligand 10 (CXCL10), CXCL1, C-C motif chemokine ligand 12 (CCL12), CCL3, TNFα, and IL-6. The addition of endotoxin (LPS) triggered the release of chemokines and cytokines above baseline levels (untreated) (Fig. 2D), which was verified at the transcriptional level, with an increase in the mRNA expression of iNOS, TNF-α, IL-1β, IL-6, and CCL3 (Fig. 2E), showing that changes in RNA translated into changes in protein levels. Therefore, NEL-MG acted as a substrate for studying the functional changes in microglia to screen for inflammatory modulators.

Long-term passage culture of NEL and a higher expression of NEL-MG on microglia signature genes
After validating the microglial function of NEL-MG, we analyzed whether the long-term passage culture of E13.5 NEL was possible. Whenever the cells reached stratification, the cells in one 25 T flask were passage cultured into two 25 T flasks. As we undertook this passage culture, we obtained approximately double the microglial cells until at least passages (P) 6-7 (first obtained microglial cell number × 2 n , n = passage number). This was because we could obtain about 50% of CD11b-positive cells from NEL, despite the long-term passage culture ( Fig. 3A and Additional file 1: Fig. S3). However, the required time for the next passage gradually increased as the passageculture progressed and the period without obtaining twice as many cells only occurred after passage 7. Therefore, the total number of microglia obtained when we used a cutoff of P7 (100 days) was 30 times higher than using the neonatal microglia method (Additional file 1: Fig. S3). We next examined the alterations in the microglial signature genes in NEL-MG obtained from longterm cultured NEL to determine the optimal period to mimic adult microglia. NEL-MG were obtained by isolation from P0 (21 days), P1 (30 days), P2 (40 days), P3 (50 days), P4 (60 days), P7 (100 days), P8 (120 days), and P10 (180 days) of NEL culturing, and the expression of microglial signature genes in each NEL-MG group was compared to that in neonatal microglia. The expression of most of the microglia identity genes was stably maintained and was higher than in neonatal microglia up to P10 ; however, the expression levels of Tgfb1, Mafb, Trem2, and Csf1r were significantly lower at P8 than P0 (Fig. 3B). The expression level of the representative adult microglia gene, Tmem119, gradually increased until isolation from the P3 NEL and decreased afterward; however, it remained approximately threefold higher than Tmem119 expression in neonatal microglia. Our data strongly suggest that adult microglia-like NEL-MG were obtainable from cultured NEL for P0-P7 (about 21-100 days). However, the IR of microglial markers, such as IBA-1, CX3CR1, and TMEM119, and their phagocytic function did not change, regardless of the NEL culture duration (Fig. 3C and D).

Banked NEL-MG stably maintain the expression of overall microglia signature genes
Animals must be sacrified whenever we need to obtain primary microglia; therefore, whether NEL is bankable needs to be determined. NELs (1 × 10 7 cells/mL) were banked for approximately 10 months in media composed of Dulbecco's modified Eagle medium (DMEM) with 20% fetal bovine serum (FBS) and 10% DMSO (banked NEL, bNEL). As shown in Fig. 4A, after thawing, the bNEL proliferated well, similar to fresh NEL. To investigate whether the banking timing affected NEL-MG characteristics, NELs were banked at different passages (P1, P2, P3, and P4) and were cultured for 70 days after thawing. Each NEL-MG group isolated from bNEL at different time points (7,14,21,40, and 70 days) showed a stable expression of microglia signature genes. The expression levels of these genes remained higher than those in neonatal microglia (Fig. 4B); however, TMEM119 mRNA expression showed a decreasing trend at 70 days (not significant). Csf1r expression was also reduced after thawing; however, its mRNA level was gradually restored as the culture progressed. NEL-MG isolated from bNEL did not show alterations in the phagocytic function compared to NEL-MG isolated from fresh NEL ( Fig. 3D and C).

Transcriptome analysis revealed that NEL-MG are closer to adult microglia than neonatal microglia
To more thoroughly characterize NEL-MG compared to the BV2 cell line, neonatal microglia, and adult microglia, we conducted transcriptome analysis. Using The PCA Euclidean distances to the adult microglia were calculated between all pairs of points in each group, using PC1, PC2, and PC3 on the microglia signature genes (Additional file 1: Table S1) [8,16]. NEL-MG and bNEL-MG were significantly closer to adult microglia than BV2 and neonatal microglia (Fig. 5B). The hierarchy analysis of the microglia signature genes showed that NEL-MG and bNEL-MG formed a cluster and had more similarities to adult microglia than to BV2 cell line and neonatal microglia (Fig. 5C). The overall expression changes (z-score normalized) in microglia signature genes were compared among the BV2 cell line, neonatal microglia, NEL-MG, and adult microglia (mean ± standard deviation [SD]). Both NEL-MG and adult microglia were localized in the opposite area (positive Z-score) from neonatal microglia and the BV2 cell line (negative Z-score). The overall expression of microglia signature genes in the NEL-MG was elevated to the same expression level as that of the genes in adult microglia (Fig. 5D). In a normalized gene expression plot, the scatters in NEL-MG were more overlapped and closer to the diagonal line than neonatal microglia (Additional file 1: Fig. S4). We next performed differential gene expression analysis (DEG, absolute fold changes > 1.5, p < 0.05) and identified that NEL-MG shared 789 genes (up DEG: 302, down DEG: 487) with adult microglia (Fig. 5E). Gene ontology (GO) analysis revealed the gene subsets of the immune system process, actin filament organization, intracellular signaling transduction, and innate immune response (Fig. 5F). The KEGG pathway of the shared genes indicated Fc gamma R-mediated phagocytosis, leukocyte transendothelial migration, and chemokine signaling pathway (Fig. 5G). NEL-MG showed 2782 DEG (2,057 upregulated and 725 downregulated) compared to neonatal microglia (Fig. 5H), and the GO analysis revealed gene subsets, including those involved in endocytosis, cell adhesion, cell migration, cholesterol biosynthetic process, cholesterol metabolic process, and the negative regulation of cell proliferation (Fig. 5I). To better understand the differential expression level of microglia signature genes between NEL-MG and neonatal microglia, we compared the fold change of the expression of microglia signature genes (Additional file 1: Table S1), and 18 genes were significantly changed (Fig. 5J).

Comparison of NEL-MG with adult microglia regarding microglia signature genes
To further confirm the transcriptome analysis, we compared the mRNA expression of microglial signature genes among the BV2 cell line, SIM-A9 cell line, neonatal microglia, NEL-MG (P3, 50 days), and acutely isolated adult microglia. qRT-PCR analyses demonstrated that NEL-MG had higher or similar expression levels of microglia signature genes, including Tgfb1, Tgfbr1, Trem2, Csf1r, C1qa, Pros1, and Gas6 (Fig. 6A). Although Tmem119 and P2ry12 expression levels of NEL-MG did not reach those in adult microglia, they were higher than in neonatal microglia. In addition, the intensity of TMEM119 IR in NEL-MG (P3, 50 days) was similar to that in adult microglia, whereas BV2 and neonatal microglia did not show TMEM119 IR (Fig. 6B).

Cultured NEL-MG showed re-expansive capacity on neuroepithelial cells
To further explore whether cultured NEL-MG could be proliferative again on neuroepithelial cells as a feeder, (See figure on next page.) Fig. 3 Stable maintenance and superiority of NEL-MG on adult microglial genes despite the long-term passage culture. When NEL reached stratification, cells in one 25 T Flask were divided into two 25 T flasks, and then cultured for 21 days (d) for flow cytometry. A The ratio of CD11b + microglia among NEL was approximately 50%, showing a stable maintenance ratio, despite the long-term passage culture (n = 3, mean ± SD) in flow cytometry. B NEL-MG were isolated based on the NEL passage culture (P0, P1, P2, P3, P4, P7, P8, and P10) and alterations in the adult microglial genes were examined compared to neonatal microglial cells. The experiment was performed three times independently (n = 3 per group) and the data represent mean ± standard error of the mean (SEM). A post hoc test was conducted using Dunnett's multiple comparison test. *p < 0.05, ***p < 0.0001, ****p < 0.00001 compared to the 21-d group. Gray zone represents a value = 1 (neonatal microglia). we isolated and cultured NEL-MG derived from NEL of GFP mice (GFP-expressing NEL-MG). Then, the cultured GFP-expressing NEL-MG were transferred on the remaining neuroepithelial cells (from B6 mice) after NEL-MG isolation. As shown in Fig. 7A and B, using flow cytometry, although the relative ratio of GFP-expressing NEL-MG did not increase significantly over time, we could confirm that GFP + Ki67 + cells were gradually increased in the immunofluorescence study. Therefore, already cultured NEL-MG could have a proliferative capacity when they were plated again on neuroepithelial cells.

Tracking of NEL-MG within neurospheroids
Taking advantage of the high cell yield, we established CellTracker-tagging NEL-MG (red) and mixed them with neurospheroids (NS) at different ratios and times. NEL-MG were mixed evenly with NS when we co-mixed them (Additional file 1: Fig. S5). We could easily detect red color of NEL-MG within NS.

Discussion
Our robust protocol allowed us to obtain bankable and expandable microglial cells, leading to the production of a high yield of microglial cells that were closer to adult microglia than neonatal microglia based on PCA. We based our study on the developmental process, in which mouse microglial cells were derived from yolk sac macrophage precursors and colonized into nascent parenchyma via the head neuroepithelial layer between E8.5 and E18.5 [1]. The neuroepithelial layer in this area includes mainly two types of cells: neuroepithelial cells and microglia progenitors. Thus, we obtained microglial cells by dissecting and culturing the neuroepithelial layer cells with lower cell heterogeneity than the brain cortex, which is composed of neurons, microglia, astrocytes, and oligodendrocytes, making it possible to obtain purer microglial cells. Neuroepithelial cells were assumed to act as feeders for microglia proliferation and maturation, which was evidenced by our data, showing that microglia progenitors proliferate with Ki67-positive labeling on neuroepithelial cells, although NEL-MG alone failed to proliferate. Therefore, neuroepithelial cells within a co-culture system play a critical role as feeders for microglial proliferation and maturation. This result also indicates that the alone cultured NEL-MG can have proliferative capacity again as microglia progenitors by the neuroepithelial support. Similarly, an astrocyte feeder layer or an astrocyte-conditioned medium makes microglia morphology and other properties resemble adult microglia more closely [17,18].
Compared to the astrocyte feeder support, our protocol has advantages in a simpler methodology (dissection and culture of NEL at E13.5) without independent preparation process of feeder cells. In addition, NEL was bankable and passage culture of NEL was possible, resulting in high yields of microglia obtainment. Microglia rely on sustained CSF-1 stimuli, which is a well-established trophic cue for survival [19], and TGF-β family members promote microglial survival and development [8]. Along with CSF-1 and TGFβ, cholesterol is another main factor that permits the robust survival of highly ramified adult microglia [17]. Our GO results indicate that NEL-MG shared pathways in general microglia function with adult microglia compared to neonatal microglia. However, NEL-MG was distinct from neonatal microglia, especially regarding the cholesterol biosynthetic process and metabolic process. Given the link between an inadequate delivery of cholesterol and generalized microglial dysfunction, the cholesterol metabolic system might be involved in key features in a more matured form of NEL-MG than neonatal microglia. The expression of microglial signature genes, including TMEM119, P2RY12, and CX3CR1, is gradually reduced with age or in neurodegenerative diseases, suggesting the loss of homeostatic microglial function [20]. De novo synthesized cholesterol transported via APOE-or APOJ-containing nanodiscs within the CNS [21,22] contribute to amyloid β clearance and microglial cholesterol content [23]. Increased lipid metabolism is required to fuel protective cellular functions, such as phagocytosis [24]. Thus, we speculate that alterations in the cholesterol metabolic system might be associated with the characteristics of the matured form of NEL-MG, which is closer to adult microglia.
There are several aspects that require further exploration regarding NEL-MG. First, we need to clarify how neuroepithelial cells make microglia progenitor to be mature and proliferate. Like astrocyte-derived factors, neuroepithelial cells might play a role for microglia survival, maturation, and proliferation. Mature microglia require continuous signaling from the healthy CNS [17]. Neuroepithelial cells function as stem cells and can differentiate to astrocyte, oligodendrocytes, and neuronal precursors, suggesting that neuroepithelial cells have a potential that mimic CNS microenvironment [25]. Thus, we speculate that various trophic factors including abovementioned defined factors such as GM-CSF, IL-34, TGF-β, and cholesterol might be produced from neuroepithelial cells. Second, the supportive capacity of neuroepithelial cells as a feeder gradually decreased as the passage culture of NEL progressed, leading to a decrease in the microglial proliferative capacity. Third, the expression of hexosaminidase subunit beta (Hexb), which is a microglial core gene stably expressed despite the pathological status [26], and colony stimulating factor receptor 1 (Csf1r) was reduced in bNEL-MG compared to pre-banked NEL-MG and there was no difference in Hexb expression between NEL-MG and neonatal microglia. To overcome these limitations, further studies are required to develop an advanced methodology to produce improved NEL-MG with defined factors, which can induce sustained proliferation, maturation, passage culture, and banking, instead of using neuroepithelial cell feeder.
Regarding the applications of our method, we propose the following as a platform for future use in the neuroscience field: (1) mass production of microglial cells for high-throughput drug screening, and (2) rapid and efficient generation of mutant microglial cells. Compared to neonatal microglia culture, our proposed method enabled us to obtain approximately 30 times more microglial cells when we used a cutoff of 100 culture days. Drug screening for inflammatory modulators targeting neuroinflammation requires significant amounts of microglial cells; however, the currently used in vitro method for primary microglia can barely meet the requirements due to limited cell yields and the number of animals required. Thus, our method contributes to overcoming the main limitation of the currently used technique. In addition, the introduction of a newly reported mutant gene into the NEL allowed the production of mass mutant NEL-MG. Using this methodology, we can rapidly evaluate the functional characteristics of mutant microglia without using a mutant animal generation. Given that the in vivo brain exhibits complex cell-cell interactions supported by contact-dependent signaling from the surrounding cells, mutant NEL-MG might be mixed with a brain organoid to further observe their behavior in a 3D brain environment, based on the fact that NEL-MG is mixed evenly with neurospheroids (Additional file 1: Fig. S4) [12]. In addition, NEL-MG might contribute to the development of a brain organoid with a controllable microglia ratio because the currently used brain organoid does not contain microglial cells, although one study has reported that a brain organoid might innately contain microglial cells [27]. Overall, our proposed method contributes to 3D brain culture and conventional 2D primary microglial culture.

Conclusions
In conclusion, we expect that our methodology contributes to overcoming the limitations of previous in vitro culture methods for microglia study by leveraging the NEL-MG platform, and increasing the adult-like microglial cell yield. Above all, we believe that our new methodology will reduce dramatically the use of experimental animals and increase the accessibility of microglial research. between all pairs of points in each group using PC1, PC2, and PC3 on microglial signature genes. **** p < 0.00001. C Clustering heatmap representation of microglia signature gene expression between clusters. The scale represents the median absolute deviation by row. For calculating the distance, a Euclidean distance metric and average linkage clustering were performed using MeV software. D The overall expression changes (z-score normalized) were plotted among groups as mean ± SD. E Differentially expressed genes between NEL-MG and adult MG. The number of shared genes was 789. F Gene ontology (GO) analysis of the shared genes (789 genes). G KEGG pathway analysis of the shared genes (789 genes).